Antiviral agents

ABSTRACT

An antiviral agent is provided, having a phosphorodiamidate morpholino oligomer with an antisense sequence to a portion of a genome of a strain of Zika virus (ZIKV). The antiviral agent finds many uses, such as in a pharmaceutical composition, a method of treating ZIKV-mediated disease, a method of preventing ZIKV-mediated disease, a method of reducing or preventing the replication of ZIKV in a host cell, a method of controlling the spread of ZIKV in donated tissue, a treated tissue sample, and in the manufacture of a medicament for the treatment or prevention or ZIKV-mediated disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of, and cites the priority of,PCT/US2017/066270 filed 14 Dec. 2017, which is currently pending, andcites the priority of U.S. Patent Application Nos. 62/434,802 (filed on15 Dec. 2016) and 62/560,144 (filed on 18 Sep. 2017). All of theforegoing applications are incorporated by reference herein in theirentireties.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The material in the accompanying sequence listing is hereby incorporatedby reference in its entirety into this application. The accompanyingfile, named Sequences_212149_401017.txt, was created on andelectronically submitted via EFS-Web on Jun. 28, 2021 and is 45.6 KB.

BACKGROUND A. Field of the Disclosure

The present disclosure relates generally to medicine, and specificallyto antiviral agents. Such agents as well as methods and kits for usetherewith are provided.

B. Background

Zika virus (ZIKV) is a member of the Flaviviridae family, genusFlavivirus, which also includes Dengue, West Nile, Japaneseencephalitis, and yellow fever viruses. ZIKV is a mosquito-bornearbovirus transmitted primarily by vectors from the Aedes family, inparticular Aedes aegypti and Aedes albopictus. ZIKV has quickly spreadto more than 50 countries in the Americas and the Caribbean, infectingmore than 2 million people. Infection with ZIKV results in asymptomaticdisease in 70%-80% of infected individuals; however, ZIKV infection hasbeen strongly associated with increased incidence of Guillain-Barrésyndrome and microcephaly in infants. Clinical presentations of ZIKVinfection include skin rash, headache, myalgia, joint pain, andconjunctivitis, but is largely self-limiting. However, ZIKV disease inthe context of immunosuppression is poorly understood. In a small casestudy by Nogueira et al., they find that allograft transmission of ZIKVcan occur in immunosuppressed SOTp (solid organ transplant patients)resulting in clinical disease in both renal and liver transplantpatients. In this study, at admission the four patients infected withZIKV after transplantation presented with bacterial infection, fever,myalgia, and adynamia along with signs of acute liver or renal damage.They did not have a rash, conjunctivitis, or neurological symptoms, butthree of four were anemic and all were thrombocytopenic.

Currently there is no specific treatment or vaccine for ZIKV infection.This represents an urgent unmet medical need for efficacioustherapeutics for ZIKV. Even if a vaccine were to be developed, sporadicoutbreaks of ZIKV disease could warrant widespread vaccination that maynot be cost effective. The need for therapeutic interventions to treatacute disease or timely prophylaxis for immunosuppressed SOTp receivingan allograft from a ZIKV infected donor is essential.

SUMMARY

The problems expounded above, as well as others, are addressed by theinvention of an antiviral agent that effectively prevents thereplication of ZIKV (although it is to be understood that not all suchproblems will be addressed by every such embodiment).

In a first aspect, an antiviral agent is provided, comprising aphosphorodiamidate morpholino oligomer comprising an antisense sequenceto a portion of a genome of a strain of ZIKV.

In a second aspect, a pharmaceutical composition for the treatment orprevention of a disease mediated by ZIKV is provided, the compositioncomprising: the antiviral agent above and a pharmaceutically acceptablecarrier.

In a third aspect, a method of treatment or prevention of a diseasemediated ZIKV in a subject in need thereof is provided, the methodcomprising administering to the subject a therapeutically effectiveamount of the pharmaceutical composition above.

In a fourth aspect, a method of reducing or preventing the replicationof ZIKV in a host cell is provided, the method comprising contacting thehost cell with the antiviral agent above.

In a fifth aspect, a method of controlling the spread of ZIKV in donatedtissue is provided, the method comprising exposing the donated tissue toan effective amount of the agent above.

In a sixth aspect, a treated donated tissue sample is provided,comprising a sample of donated tissue and the agent above.

In a seventh aspect, a use of the agent above in the manufacture of amedicament for the treatment or prevention of a disease mediated by ZIKVis provided.

The above presents a simplified summary in order to provide a basicunderstanding of some aspects of the claimed subject matter. Thissummary is not an extensive overview. It is not intended to identify keyor critical elements or to delineate the scope of the claimed subjectmatter. Its sole purpose is to present some concepts in a simplifiedform as a prelude to the more detailed description that is presentedlater.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B: RT-PCR analysis of human glomerular podocytes infectedwith ZIKV after treatment with DWK-M1. (1A) Quantitative real-timeqRT-PCR analysis of glomerular podocytes infected with ZIKV for 72 h.Shown are mock infected podocytes, podocytes infected with wildtype ZIKVand podocytes pretreated for 24 h with the DWK-M1 morpholino+ZIKV for 72h. (1B) qRT-PCR analysis of glomerular podocytes infected with ZIKV for72 h. Shown are mock infected podocytes, podocytes exposed to a controlmorpholino (Co DWK), podocytes exposed to Co DWK+ZIKV infected,podocytes exposed to DWK-M1 only, podocytes infected with wildtype ZIKV,and podocytes exposed to DWK-M1+ZIKV infected. All values werenormalized to GAPDH.

FIG. 2: Immunofluorescent staining of ZIKV infected podocytes using the4G-2 antibody to the E-protein of ZIKV, (A) mock infected podocytesstained with 4G-2 antibody (B) podocytes infected with wildtype ZIKV for72 h and stained with the 4G-2 antibody (C) podocytes pretreated withDWK-M1 for 24 h then infected with ZIKV for 72 h and stained with the4G-2 antibody. Phase and fluorescent images were taken on a NikonTE2000S microscope mounted with a charge-coupled device (CCD) camera at200× magnification. For fluorescent images,4′,6-diamidino-2-phenylindole (DAPI) was used to stain the nuclei blue.

FIG. 3: Shown is a western blot analysis of protein lysates from ZIKVinfected podocytes. Results include mock infected podocytes, podocytesexposed to a control morpholino (Co DWK), podocytes exposed to the CoDWK and ZIKV infected, podocytes exposed to DWK-M1 alone, podocytesinfected with wildtype ZIKV, and podocytes exposed to DWK-M1+ZIKVinfected. The ZIKV expression of the E protein (E2 antigen) is shown inthe top panel. The middle panel shows the podocyte marker Synaptopodinand bottom panel shows GAPDH as a loading control.

FIG. 4A: Real time PCR analysis of podocytes infected with ZIKV afterexposure to DWK-M1 72 hours after infection for RANTES. Results showZIKV induction of RANTES in podocytes after 24 h pretreatment with theCo DWK, and the DWK-M1 morpholinos. Mock infected and morpholinocontrols without ZIKV are also shown. All values were normalized toGAPDH.

FIG. 4B: Real time PCR analysis of podocytes infected with ZIKV afterexposure to DWK-M1 72 hours after infection for MP1-alpha. Results showZIKV induction of MIP-1alpha after 24 h pretreatment with the Co DWK,and the DWK-M1 morpholinos. Mock infected and morpholino controlswithout ZIKV are also shown. All values were normalized to GAPDH.

FIG. 4C: Real time PCR analysis of podocytes infected with ZIKV afterexposure to DWK-M1 72 hours after infection for TNFα. Results show ZIKVinduction of TNF-alpha in podocytes after 24 h pretreatment with the CoDWK, and the DWK-M1 morpholinos. Mock infected and morpholino controlswithout ZIKV are also shown. All values were normalized to GAPDH.

FIG. 4D: Real time PCR analysis of podocytes infected with ZIKV afterexposure to DWK-M1 72 hours after infection for IFN-b. Results show ZIKVinduction of INF-b in podocytes after 24 h pretreatment with the Co DWK,and the DWK-M1 morpholinos. Mock infected and morpholino controlswithout ZIKV are also shown. All values were normalized to GAPDH.

FIG. 5: Schematic structure of a Vivo-morpholino. A Vivo-morpholino iscomposed of a 25-mer long morpholino oligonucleotide covalently linkedto an octa-guanidine dendrimer, which serves as a delivery moiety. Anucleotide sequence of ZIKV PRVABC59 Vivo-morpholino DWK-1 (previouslyreferred to as DWK-M1) is shown).

FIG. 6: Dose-dependent effect of DWK-1 and Co DWK-1 on the accumulationof intracellular ZIKV RNA in infected human podocytes. Podocytes werepretreated for 24 h with the indicated doses of DWK-1 or Co DWK-1,rinsed and infected with ZIKV at MOI of 0.1 in the absence ofmorpholinos. Total RNA was isolated from the mock infected andZIKV-infected cells at 72 h p.i. Expression of ZIKV RNA was determinedby qRT-PCR and normalized to GAPDH mRNA expression. ZIKV infections wereperformed in triplicate. Values represent mean±SD.

FIG. 7: Mortality of CD-1 mice 96 h after subcutaneous injection ofDWK-1.

FIGS. 8A-8D: DWK-1 reduces ZIKV RNA genome copy number in infectedpodocytes. (8A) Ten-fold dilutions of synthetic ZIKV RNA (VR-3252SD, 10⁶to 10 copies) were amplified by qRT-PCR using ZIKV specific primers.Amplification curves are shown. NTC, no template control. (8B) Theregression line was established by plotting the threshold cycles (CT)values against the copy number of synthetic RNA. The coefficient ofdetermination R² was 0.997 and slope was −3.923. (8C) Total cellular RNAisolated from mock, ZIKV infected cells, cells pretreated for 24 h with10 μM DWK-1 or Co DWK-1 alone, or cells pretreated with morpholinos andinfected with ZIKV for 48 h was analyzed by qRT-PCR for the expressionof ZIKV and GAPDH RNA. Relative expression of intracellular ZIKV RNA wasnormalized to GAPDH mRNA. Values represent mean±SD of 3 independentsamples. *P<0.001. (8D) Quantitation of ZIKV genome copy number in totalintracellular RNA prepared as described in (8C) shows 94.1% reduction inZIKV copy number in infected cells pretreated with DWK-1. Valuesrepresent mean±SD of 3 independent samples. *P<0.001. ND, not detected.

FIGS. 9A-9D: Immunofluorescent staining of ZIKV infected podocytes usingthe 4G-2 antibody specific to the E protein of ZIKV. (9A) Mock infectedpodocytes stained with 4G-2 antibody, (9B) Podocytes infected withwildtype ZIKV for 72 h and stained with the 4G-2 antibody, (9C)Podocytes pretreated with DWK-1 for 24 h, rinsed and infected with ZIKVfor 72 h were stained with the 4G-2 antibody. (9D) Isotype control forthe 4G-2 antibody. Fluorescent images were taken on a Nikon TE2000Smicroscope mounted with a charge-coupled device (CCD) camera at 200×magnification. DAPI (4′,6-diamidino-2-phenylindole) was used to stainthe nuclei blue.

FIG. 10: DWK-1 inhibits expression of E protein in ZIKV-infectedpodocytes. Western blot analysis of protein lysates from uninfected andZIKV infected podocytes. Control protein lysates were prepared from mockinfected podocytes and podocytes pretreated for 24 h with 10 μM DWK-1 orCo DWK-1, rinsed and cultured for additional 72 h without addedmorpholinos. Untreated podocytes or cells pretreated for 24 h with DWK-1or Co DWK-1 were subsequently infected with ZIKV and protein lysateswere prepared 72 h after ZIKV infection. The ZIKV expression of the Eprotein (E2 antigen) is shown in the top panel. The middle panel showsthe podocyte biomarker Synaptopodin and the bottom panel shows GAPDH asa loading control.

FIGS. 11A-11F: DWK-1 inhibits ZIKV-induced proinflammatory cytokine geneexpression. Podocytes were pretreated for 24 h with 10 μM DWK-1 or CoDWK-1 and infected with ZIKV at MOI 0.1. Mock infected cells and cellstreated only with DWK-1 or Co DWK-1 were included as controls. Total RNAwas isolated at 72 h p.i. and indicated cytokine gene expression wasquantitated by qRT-PCR and normalized to GAPDH mRNA. Results show theeffect of DWK-1 and Co DWK-1 on the expression of selected cytokinegenes in ZIKV infected podocytes: (11A) IFN-β, *P<0.001 (11B) RANTES,*P<0.001 (11C) MIP-1α, *P<0.005 (11D) TNF-α, **P<0.01 (11E) IL-1α,*P<0.01, and (11F) IL-6, ns (statistically not significant). Valuesrepresent mean±SD of 3 independent samples. The expression of cytokinegenes mRNA in mock infected cells was set as 1.0.

FIG. 12: Schematic presentation of flavivirus (ZIKV) highly structured5′UTR and 3′UTR. Highly conserved sHP-3′SL region is targeted by DWK-2(box). ORF, open reading frame coding for virus polyprotein

FIG. 13: A sequence in the sHP-3′SL region of the 3′UTR of ZIKV strainsthat is targeted by DWK-2 morpholino is shown in white-on-black.Sequence alignment for two different ZIKV strains is shown.

FIGS. 14A-14D: DWK-2 inhibits ZIKV-induced proinflammatory cytokine geneexpression. Podocytes were pretreated for 24 h with 10 μM DWK-2 or CoDWK-1 (control) and infected with ZIKV. Mock infected cells and cellstreated only with DWK-2 or Co DWK-2 were included as controls. Total RNAwas isolated at 72 h p.i. and intracellular ZIKV RNA was quantitated byqRT-PCR and normalized to GAPDH mRNA levels. Results show inhibitoryeffect of DWK-2 on the expression of ZIKV induced (14A) IL-6, (14B)IL-1α, (14C) INF-β, (14D) RANTES genes. Values represent mean±SD of 3independent samples. The expression of cytokine genes mRNA in mockinfected cells was set as 1.0.

FIGS. 15A-15B: (15A) DWK-2 inhibits accumulation of intracellular ZIKVRNA in infected podocytes. Podocytes were pretreated for 24 h with 10 μMDWK-2 or Co DWK-2 (control) and infected with ZIKV. Mock infected andDWK-2 and Co DWK-2 pretreated cells were included as controls. Total RNAwas isolated at 72 h p.i. and intracellular ZIKV RNA expression wasdetermined by qRT-PCR and normalized to GAPDH mRNA levels. ZIKVinfections were performed in triplicate. Values represent mean±SD of 3independent samples. ND, not detected. (15B) DWK-2 reduces ZIKV RNAgenome copy number in infected podocytes. Total cellular RNA isolatedfrom mock, ZIKV infected cells, or cells pretreated for 24 h with 10 μMDWK-2 alone, or from DWK-2 pretreated cells and infected with ZIKV for48 h was analyzed by qRT-PCR for the expression of ZIKV and GAPDH RNA.Relative expression of intracellular ZIKV RNA normalized to GAPDH RNA isreduced by 94.2%. Quantitation of ZIKV genome copy number in totalintracellular RNA shows a reduction in ZIKV copy number in infectedcells pretreated with DWK-2. Values represent mean±SD of 3 independentsamples. ND, not detected.

DETAILED DESCRIPTION A. Definitions

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art of this disclosure. It will be furtherunderstood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the specification andshould not be interpreted in an idealized or overly formal sense unlessexpressly so defined herein. Well known functions or constructions maynot be described in detail for brevity or clarity.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. As used herein, thesingular forms “a”, “an”, and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.

The terms “first”, “second”, and the like are used herein to describevarious features or elements, but these features or elements should notbe limited by these terms. These terms are only used to distinguish onefeature or element from another feature or element. Thus, a firstfeature or element discussed below could be termed a second feature orelement, and similarly, a second feature or element discussed belowcould be termed a first feature or element without departing from theteachings of the present disclosure.

The term “consisting essentially of” means that, in addition to therecited elements, what is claimed may also contain other elements(steps, structures, ingredients, components, etc.) that do not adverselyaffect the operability of what is claimed for its intended purpose asstated in this disclosure. This term excludes such other elements thatadversely affect the operability of what is claimed for its intendedpurpose as stated in this disclosure, even if such other elements mightenhance the operability of what is claimed for some other purpose.

The terms “about” and “approximately” shall generally mean an acceptabledegree of error or variation for the quantity measured given the natureor precision of the measurements. Typical, exemplary degrees of error orvariation are within 20 percent (%), preferably within 10%, and morepreferably within 5% of a given value or range of values. For biologicalsystems, the term “about” refers to an acceptable standard deviation oferror, preferably not more than 2-fold of a given value. Numericalquantities given herein are approximate unless stated otherwise, meaningthat the term “about” or “approximately” can be inferred when notexpressly stated.

Terms such as “administering” or “administration” include acts such asprescribing, dispensing, giving, or taking a substance such that what isprescribed, dispensed, given, or taken actually contacts the patient'sbody externally or internally (or both). In embodiments of thisdisclosure, terms such as “administering” or “administration” includeself-administering, self-administration, and the like, of a substance.Indeed, it is specifically contemplated that instructions or aprescription by a medical professional to a subject or patient to takeor otherwise self-administer a substance is an act of administration.

The terms “prevention”, “prevent”, “preventing”, “suppression”,“suppress”, and “suppressing”, as used herein, refer to a course ofaction (such as administering a pharmaceutical composition) initiatedprior to the onset of a clinical manifestation of a disease state orcondition so as to reduce its likelihood or severity. Such reduction inlikelihood or severity need not be absolute to be useful.

The terms “treatment”, “treat”, and “treating”, as used herein, refer toa course of action (such as administering a pharmaceutical composition)initiated after the onset of a clinical manifestation of a disease stateor condition so as to eliminate or reduce such clinical manifestation ofthe disease state or condition. Such treating need not be absolute to beuseful.

The term “in need of treatment”, as used herein, refers to a judgmentmade by a caregiver that a patient requires or will benefit fromtreatment. This judgment is made based on a variety of factors that arein the realm of a caregiver's expertise, but that include the knowledgethat the patient is ill, or will be ill, as the result of a conditionthat is treatable by a method or device of the present disclosure.

The term “in need of prevention”, as used herein, refers to a judgmentmade by a caregiver that a patient requires or will benefit fromprevention. This judgment is made based on a variety of factors that arein the realm of a caregiver's expertise, but that include the knowledgethat the patient will be ill or may become ill, as the result of acondition that is preventable by a method or device of the disclosure.

The terms “individual”, “subject”, or “patient”, as used herein, referto any animal, including mammals, such as mice, rats, other rodents,rabbits, dogs, cats, swine, cattle, sheep, horses, primates, and humans.The term may specify male or female or both, or exclude male or female.

The term “therapeutically effective amount” (or simply “effectiveamount”), as used herein, refers to an amount of an agent, either aloneor as a part of a pharmaceutical composition, that is capable of havingany detectable, positive effect on any symptom, aspect, orcharacteristics of a disease state or condition. Such effect need not beabsolute to be beneficial.

The term “pharmaceutically acceptable salts”, as used herein, includessalts of the antiviral agents which are prepared with relativelynontoxic acids or bases, depending on the particular substituents foundon the compounds described herein. When compounds of the presentinvention contain relatively acidic functionalities, base addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogen carbonic, phosphoric,monohydrogen phosphoric, dihydrogen phosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as thesalts derived from relatively nontoxic organic acids like acetic,propionic, isobutyric, oxalic, maleic, malonic, benzoic, succinic,suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic,citric, tartaric, methanesulfonic, and the like. Also included are saltsof amino acids such as arginate and the like, and salts of organic acidslike glucuronic or galactunoric acids and the like (see, for example,Berge, S. M., et al., “Pharmaceutical Salts”, Journal of PharmaceuticalScience, 1977, 66, 1-19). Certain specific compounds of the presentinvention contain both basic and acidic functionalities that allow thecompounds to be converted into either base or acid addition salts.

Nucleic acids are “complementary” to each other, as used herein, when anucleotide sequence in one strand of a nucleic acid, due to orientationof its nucleotide hydrogen atoms, hydrogen bonds to another sequence onan opposing nucleic acid strand (of course, a strand of a nucleic acidmay be self-complementary as well). The complementary bases typicallyare, in DNA, A with T, and C with G, and, in RNA, C with G, and U withA. Complementarity can be perfect or substantial/sufficient. Perfectcomplementarity between two nucleic acids means that the two nucleicacids can form a duplex in which every base in the duplex is bonded to acomplementary base by Watson-Crick pairing. “Substantial” or“sufficient” complementarity means that a sequence in one strand is notperfectly complementary to a sequence in an opposing strand, but thatsufficient bonding occurs between bases on the two strands to form astable hybrid complex at a given set of hybridization conditions (e.g.,salt concentration and temperature). Such conditions can be predicted byusing the sequences and standard models to predict the T_(m) ofhybridized strands, or by empirical determination of T_(m) by usingestablished methods. T_(m) refers to the temperature at which apopulation of hybridization complexes formed between two nucleic acidstrands are 50% denatured. At a temperature below the T_(m), formationof a hybridization complex is favored, whereas at a temperature abovethe T_(m), melting or separation of the strands in the hybridizationcomplex is favored. Such stringency is based on the melting temperature(T_(m)) of the nucleic acid binding complex, as taught in Berger andKimmel (1987, Guide to Molecular Cloning Techniques, Methods inEnzymology, 152, Academic Press, San Diego Calif.). The T_(m) of anannealed duplex depends on the base composition of the duplex, thefrequency of base mismatches, and the ionic strength of the reactionmedium. The T_(m) of a duplex can be calculated by those of ordinaryskill in the art based on these two factors using accepted algorithms.Maximum stringency typically occurs at about 5° C. below T_(m); highstringency at about 5-10° C. below T_(m); intermediate stringency atabout 10-20° C. below T_(m); and low stringency at about 20-25° C. belowT_(m). As will be understood by those of skill in the art, a maximumstringency hybridization can be used to identify or detect identicalnucleotide sequences while an intermediate (or low) stringencyhybridization can be used to identify or detect similar or relatedsequences. Terms such as maximally stringent, highly stringent, andpoorly stringent, refer to conditions of maximal stringency, highstringency, and low stringency respectively.

In the following discussion certain outside documents are referenced toenable the reader to make and use the subject matter described herein.Nothing contained herein is to be construed as an “admission” of priorart. Applicant expressly reserves the right to demonstrate, whereappropriate, that such documents referenced herein do not constituteprior art under the applicable statutory provisions.

B. Antiviral Agents

A phosphorodiamidate morpholino oligomer (PMO) is disclosed thatsuppresses viral replication. In the interest of clarity, not allfeatures of an actual implementation are described in thisspecification. It will of course be appreciated that in the developmentof any such actual embodiment, numerous implementation-specificdecisions must be made to achieve the worker's specific goals, such as acompliance with system-related and business-related constraints, whichwill vary from one implementation to another. Moreover, it will beappreciated that such a development effort might be complex andtime-consuming, but would nevertheless be a routine undertaking forthose of ordinary skill in the art having the benefit of thisdisclosure.

PMO are nucleic acids having conventional nucleotide bases, but abackbone of methylenemorpholine rings and phosphorodiamidate linkages.PMO bind to RNA with high specificity. This gives PMO the ability toblock the translation of mRNA by binding to complementary sequences onthe mRNA, which prevents binding of the mRNA to the ribosome.Translational blocking with PMO is highly specific, and does not resultin blocking of non-target mRNA. PMO are also much more stable than RNAand resistant to most exonucleases. An unmodified PMO has the followinggeneral structure, with each “B” being independently selected fromadenine, cytosine, guanine, or thymine:

The PMO of the agent comprises a nucleotide sequence that iscomplementary to a sequence in a viral genome (the “target sequence”).Such complementary sequence is referred to herein as the “antisensesequence”, although as explained below, in some embodiments the sequencemay deviate from an exact antisense sequence of the target. The genomemay be, without limitation, the genome of a single-stranded positivesense RNA virus, such as a flavivirus. In a specific embodiment of theagent, the genome is a genome of a strain of ZIKV. The sequence in theviral genome should be a sequence that must bind to the cellularribosome for replication to occur. This may be a sequence in astructural gene (i.e., in an open reading frame), or it may be anon-translated sequence that facilitates binding of the strand to theribosome.

For purposes of illustration, the ZIKV genome will be used as anexample. The ZIKV genome comprises an untranslated 5′ region with amethylated cap for canonical cellular translation, a single polyprotein3419 residues in length, and a non-polyadenylated 3′ region that formsstem-and-loop structures. The canonical ZIKV genome is a single-strandedRNA 10,794 base pairs (bp) long. The canonical ZIKV genome has beenassigned GENBANK accession number AY632535, and is incorporated hereinby reference in its entirety (SEQ ID NO: 1). The ZIKV genome is flankedby 5′ untranslated region (UTR) and 3′UTR. The non-coding 3′UTR ishighly structured (FIG. 12) with some regions highly conserved betweenflaviviruses. Without wishing to be bound by any hypothetical model, theinteraction between 5′ and 3′UTRs are believed to be critical for viralRNA replication. Without wishing to be bound by any hypothetical model,it is believed that RNA elements within the 3′UTR are essential forflavivirus replication and pathogenesis. Among several RNA elements inthe 3′UTR, the 3′ short hairpin structure (sHP) and 3′ stem-loop (3′SL)are highly conserved across flaviviruses and specifically ZIKV strains.

In some embodiments of the agent, the target sequence is a sequence fromthe 5′ region of the ZIKV genome, for example the region encompassingthe C (capsid) protein and the 5′ untranslated region (UTR). In aspecific embodiment of the agent, the target sequence comprises 5′-TTGGAA ACG AGA GTT TCT GGT CAT G-3′ (SEQ ID NO: 2) from the 5′ UTR. In thesame specific embodiment, the PMO comprises the sequence 5′-CAT GAC CAGAAA CTC TCG TTT CCA A-3′ (SEQ ID NO: 3). In further embodiments of theagent, the target sequence is a sequence from the 3′ region of the ZIKVgenome, for example the 3′UTR. Again turning to FIG. 12, the 3′UTR ofthe ZIKV genome contains three stem-and-loop structures (SL I, SL II,and SL III), a 3′ short hairpin structure (sHP), and a terminal 3′ endstem-and-loop structure. Various embodiments of the antiviral agenttarget one or more of these 3′ structures. The sHP is particularlyhighly conserved among strains of ZIKV (FIG. 13) (SEQ ID NOS: 26 and 27for KU955592_ZIKV and KX377335_ZIKV, respectively). A specificembodiment of the antiviral agent targets a sequence in the sHP. In aspecific embodiment of the agent, the target sequence comprises 5′-GCTGGG AAA GAC CAG AGA CTC CAT G-3′ (SEQ ID NO: 4) from the sHP. In thesame specific embodiment, the PMO comprises the sequence 5′-CAT GGA GTCTCT GGT CTT TCC CAG C-3 (SEQ ID NO: 5).

The antisense sequence will bind with high stringency to the targetsequence under physiological (intracellular) conditions. Such conditionsare understood by those of ordinary skill in the art, but will vary bycell type. For example, intracellular pH and sodium concentration variesin a narrow range by cell type. Physiological conditions for humansubjects are generally at 37° C. (98.6° F.). Typically, this means thatthe antisense sequence will have at least 80% identity with an exactcomplement of the target sequence. In various embodiments of the agentthe antisense sequence will have at least 85, 90, 95%, 96%, 97%, 98%, or99% identity with an exact complement of the target sequence. In aspecific embodiment the antisense sequence is an exact complement of thetarget sequence.

The antisense sequence will generally be about 25 bases long. This canvary somewhat, in the range of about 10-30 bases. Specific embodimentsof the antisense sequence can be any length from 10-30 bases. Morespecific embodiments are 15-25 bases. A particular embodiment ofantisense sequence is exactly 25 bases long. The PMO may compriseadditional nucleotides on the 5′ end or 3′ end (or both) of the targetrecognition sequence. In a specific embodiment, the antisense sequenceis the entire nucleotide sequence of the PMO, and there are noadditional nucleotides on the 5′ end or the 3′ end of the antisensesequence.

The PMO may have other various desirable characteristics. These mayinclude without limitation: a base sequence that has very littleself-complementarity; a high enough GC-content (guanine-cytosinecontent) (e.g. 40-60%) so that it has a high target affinity; and nostretches of four or more contiguous G to preserve water solubility.

The PMO may have modified 3′ or 5′ ends to add various additionalfunctionalities. Such modifications can include 3′ conjugation with anyof: a fluorophore, a quencher, carboxyfluorescein, lissamine, dabcyl,biotin, amine, amine with biotin, disulfide amine, pyridyl dithio,azide, and alkyne. Such modifications may include 5′ conjugation withany of: a primary amine, dabcyl, azide, and alkyne. In a specificembodiment of the agent, the PMO is modified for intracellular delivery.

Modifications for cellular delivery may include endocytosis-stimulatingpeptides, such as weak-base amphiphilic peptides taught in U.S. Pat. No.7,084,248 and commercially available under the tradename ENDO PORTERfrom Gene Tools, LLC (Philomath, Oreg., USA). In another example, thePMO is conjugated to an octa-guanidine dendrimer. A specific embodimentof the octa-guanidine dendrimer has the following structure:

C. Pharmaceutical Compositions

A pharmaceutical composition for treating or preventing a diseasemediated by ZIKV is provided, the composition comprising any of theantiviral agents provided above. The compositions disclosed may compriseone or more of such antiviral agents, in combination with apharmaceutically acceptable carrier. Examples of such carriers andmethods of formulation may be found in Remington: The Science andPractice of Pharmacy (20th Ed., Lippincott, Williams & Wilkins, DanielLimmer, editor), and are generally well understood by those skilled inthe art. To form a pharmaceutically acceptable composition suitable foradministration, such compositions will contain a therapeuticallyeffective amount of an antiviral agent.

The pharmaceutical compositions of the disclosure may be used in thetreatment and prevention methods of the present disclosure. Suchcompositions are administered to a subject in amounts sufficient todeliver a therapeutically effective amount of the antiviral agent so asto be effective in the treatment and prevention methods disclosedherein. The therapeutically effective amount may vary according to avariety of factors such as the subject's condition, weight, sex, andage. For example, some embodiments of the composition comprise up to themedian lethal dose (LD₅₀) of the antiviral agent. The LD₅₀ can beascertained using standard toxicological methods, or by reference topast studies. Alternatively, the pharmaceutical composition may beformulated to achieve a desired concentration of the antiviral agent atthe site of the infection.

The toxicity of PMO are generally very low. Embodiments of the antiviralagent have been tested for toxicity in mice (see Example 4 below). Nomortality was observed at up to 30 mg/kg. In some embodiments of thepharmaceutical composition, the PMO is administered to the subject at upto about 30 mg/kg. In further embodiments of the pharmaceuticalcomposition, the PMO is administered to the subject at up to about 5,10, 15, or 20 mg/kg. To account for possible interspecies variation insensitivity to the agent, in further embodiments of the pharmaceuticalcomposition, the PMO is administered to the subject at up to about 0.5,1, 1.5, 2, or 3 mg/kg. To further account for possible variation amongindividuals and interspecies variation, in further embodiments of thepharmaceutical composition, the PMO is administered to the subject at upto about 0.05, 0.1, 0.15, 0.2, or 0.3 mg/kg. The PMO may be administeredto the subject, such as in a pharmaceutical composition, to provide thePMO at a dosage/body mass concentration of up to an amount selectedfrom: 0.05, 0.1, 0.15, 0.2, 0.3, 0.5, 1, 1.5, 2, 3, 5, 10, 15, 20, 30mg/kg, about any of the foregoing values, and a range between any of theforegoing values.

Other factors include the mode and site of administration. Thepharmaceutical compositions may be formulated to be provided to thesubject in any method known in the art. Exemplary dosage forms includeocular, subcutaneous, intravenous, topical, epicutaneous, oral,intraosseous, intramuscular, intranasal, and pulmonary. The compositionsof the present disclosure may be formulated to be administered only onceto the subject or more than once to the subject. Furthermore, when thecompositions are administered to the subject more than once, they may beformulated for a variety of regimen, such as once per day, once perweek, once per month, or once per year. The compositions may also beformulated to be administered to the subject more than one time per day.The therapeutically effective amount of the antiviral agent andappropriate dosing regimens may be identified by testing in order toobtain optimal activity, while minimizing any potential side effects. Inaddition, formulation for co-administration or sequential administrationof other agents may be desirable.

The compositions of the present disclosure may be formulated to beadministered systemically, such as by intravenous administration, orlocally such as by subcutaneous injection or by application of a gel,fiber, paste, or cream.

The compositions of the present disclosure may further comprise agentswhich improve the solubility, half-life, absorption, etc. of theantiviral agent. Furthermore, the compositions of the present disclosuremay further comprise agents that attenuate undesirable side effectsand/or decrease the toxicity of the antiviral agent. Examples of suchagents are described in a variety of texts, such as Remington: TheScience and Practice of Pharmacy (20th Ed., Lippincott, Williams &Wilkins, Daniel Limmer, editor).

The compositions of the present disclosure can be formulated in a widevariety of dosage forms for administration. For example, thecompositions can be in the form of tablets, capsules, sachets, lozenges,troches, pills, powders, granules, elixirs, tinctures, solutions,suspensions, syrups, ointments, creams, pastes, emulsions, or solutionsfor intravenous administration or injection. Other dosage forms includefor administration transdermally, via patch mechanism or ointment.Further dosage forms include formulations suitable for delivery bynebulizers or metered dose inhalers. Any of the foregoing may bemodified to provide for timed release and/or sustained releaseformulations.

In the present disclosure, the pharmaceutical compositions may furthercomprise a pharmaceutically acceptable carrier. Such carriers mayinclude vehicles, adjuvants, surfactants, suspending agents, emulsifyingagents, inert fillers, diluents, excipients, wetting agents, binders,lubricants, buffering agents, disintegrating agents, accessory agents,coloring agents, and flavoring agents (collectively referred to hereinas a carrier). Typically, the pharmaceutically acceptable carrier ischemically inert to the antiviral agents and has no detrimental sideeffects or toxicity under the conditions of use. The pharmaceuticallyacceptable carriers can include polymers and polymer matrices. Thenature of the pharmaceutically acceptable carrier may differ dependingon the particular dosage form employed and other characteristics of thecomposition.

For instance, in compositions for oral administration in solid form,such as tablets, capsules, sachets, lozenges, troches, pills, powders,or granules, the antiviral agent may be combined with an oral, non-toxicpharmaceutically acceptable inert carrier, such as inert fillers,suitable binders, lubricants, disintegrating agents, and accessoryagents. Suitable binders include, without limitation, starch, gelatin,natural sugars such as glucose or beta-lactose, corn sweeteners, naturaland synthetic gums such as acacia, tragacanth, or sodium alginate,carboxymethylcellulose, polyethylene glycol, waxes, and the like.Lubricants used in these dosage forms include, without limitation,sodium oleate, sodium stearate, magnesium stearate, sodium benzoate,sodium acetate, and the like. Disintegrators include, withoutlimitation, starch, methyl cellulose, agar, bentonite, xanthan gum, andthe like. Tablet forms can include one or more of the following:lactose, sucrose, mannitol, corn starch, potato starch, alginic acid,microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicondioxide, croscarmellose sodium, talc, magnesium stearate, calciumstearate, zinc stearate, stearic acid, as well as the other carriersdescribed herein. Lozenge forms can comprise the active ingredient in aflavor, usually sucrose and acacia or tragacanth, as well as pastillescomprising the active ingredient in an inert base, such as gelatin andglycerin, or sucrose and acacia, emulsions, and gels containing, inaddition to the active ingredient, such carriers as are known in theart.

The composition may be also be in oral liquid form, such as a tincture,solution, suspension, elixir, and syrup; and the antiviral agents of thepresent disclosure can be dissolved in diluents, such as water, saline,or alcohols. Furthermore, the oral liquid forms may comprise suitablyflavored suspending or dispersing agents such as synthetic and naturalgums, for example, tragacanth, acacia, methylcellulose, and the like.Moreover, when desired or necessary, suitable coloring agents or otheraccessory agents can also be incorporated into the mixture. Otherdispersing agents that may be employed include glycerin and the like.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the patient, and aqueous andnon-aqueous sterile suspensions that can include suspending agents,solubilizers, thickening agents, stabilizers, and preservatives. Thecomposition may comprise a physiologically acceptable diluent, such as asterile liquid or mixture of liquids, including water, saline, aqueousdextrose and related sugar solutions, an alcohol, such as ethanol,isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol orpolyethylene glycol such as poly(ethyleneglycol) 400, glycerol ketals,such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, an oil, a fattyacid, a fatty acid ester or glyceride, or an acetylated fatty acidglyceride with or without the addition of a pharmaceutically acceptablesurfactant, such as a soap, an oil or a detergent, suspending agent,such as pectin, carbomers, methylcellulose,hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifyingagents and other pharmaceutical adjuvants.

Oils, which can be used in parenteral formulations, include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includepolyethylene sorbitan fatty acid esters, such as sorbitan monooleate andthe high molecular weight adducts of ethylene oxide with a hydrophobicbase, formed by the condensation of propylene oxide with propyleneglycol, oleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters. Suitablesoaps for use in parenteral formulations include fatty alkali metal,ammonium, and triethanolamine salts, and suitable detergents include:(a) cationic detergents such as, for example, dimethyldialkylammoniumhalides, and alkylpyridinium halides; (b) anionic detergents such as,for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether,and monoglyceride sulfates, and sulfosuccinates; (c) nonionic detergentssuch as, for example, fatty amine oxides, fatty acid alkanolamides, andpolyoxyethylene polypropylene copolymers; (d) amphoteric detergents suchas, for example, alkylbeta-aminopropionates, and 2-alkylimidazolinequaternary ammonium salts; and (e) mixtures thereof.

Suitable preservatives and buffers can be used in such formulations. Inorder to minimize or eliminate irritation at the site of injection, suchcompositions may contain one or more nonionic surfactants having ahydrophile-lipophile balance (HLB) of from about 12 to about 17.

Topical dosage forms, such as ointments, creams, pastes, and emulsions,containing the antiviral agent, can be admixed with a variety of carriermaterials well known in the art, such as, e.g., alcohols, aloe vera gel,allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristylpropionate, and the like, to form alcoholic solutions, topicalcleansers, cleansing creams, skin gels, skin lotions, and shampoos incream or gel formulations. Inclusion of a skin exfoliant or dermalabrasive preparation may also be used. Such topical preparations may beapplied to a patch, bandage, or dressing for transdermal delivery, ormay be applied to a bandage or dressing for delivery directly to thesite of a wound or cutaneous injury.

The antiviral agents of the present disclosure can also be formulated tobe administered in the form of liposome delivery systems, such as smallunilamellar vesicles, large unilamellar vesicles and antiemetics.Liposomes can be formed from a variety of phospholipids, such ascholesterol, stearylamine or phosphatidylcholines. Such liposomes mayalso contain monoclonal antibodies to direct delivery of the liposome toa particular cell type or group of cell types.

The antiviral agents of the present disclosure may also be coupled withsoluble polymers as targetable drug carriers. Such polymers can includepolyvinyl-pyrrolidone, pyran copolymer,polyhydroxypropylmethacryl-amidephenol,polyhydroxyethylaspartamidephenol, or polyethyl-eneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the antiviral agentsof the present invention may be coupled to a class of biodegradablepolymers useful in achieving controlled release of a drug, for example,polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid,polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates,and cross-linked or amphipathic block copolymers of hydrogels.

D. Methods of Use

By way of non-limiting example only, methods of using the agents andpharmaceutical compositions disclosed above are provided.

A method of treatment or prevention of a disease mediated by ZIKV in asubject in need thereof is provided, the method comprising administeringto the subject a therapeutically effective amount of any of thepharmaceutical compositions disclosed above. The disease may be any thatis caused, complicated, or exacerbated by ZIKV infection, including Zikafever, Guillain-Barré syndrome, a congenital defect, microcephaly,ocular disease, and Zika associated organ pathology. The ZIKV infectionneed not be in the subject him or herself; for example, the method couldbe used for the prevention of microcephaly in a fetus by administrationto the mother.

The method of treatment and/or prevention comprises administering to thesubject the antiviral agent in an amount sufficient to treat or preventthe ZIKV-mediated disease (therapeutically effective amount). The methodwill often further comprise identifying a subject in need of suchtreatment or prevention. Too little antiviral agent would fail toprovide the therapeutic effect. On the other hand, excessive antiviralagent could lead to undesired side effects.

The therapeutically effective amount may vary according to a variety offactors such as the subject's condition, weight, sex and age. Forexample, some embodiments of the method comprise administration of up tothe median lethal dose (LD₅₀) of the antiviral agent. The LD₅₀ can beascertained using standard toxicological methods, or by reference topast studies. Alternatively, the method may comprise delivering adesired concentration of the antiviral agent to a tissue, organ, or celltype hosts ZIKV in the subject.

If, after the administration of the antiviral agent, the subject stillhas the ZIKV-mediated disease, or is at risk for the same, then anoptional step of the method is to continue administration of theantiviral agent or pharmaceutical composition.

In one embodiment, the method comprises delivering the antiviral agentto a tissue, organ, or cell type of the subject that hosts ZIKV. Suchtissues and organs include the eye, retinal tissue, retinal endothelialcells, retinal microvascular endothelial cells, retinal pigmentedepithelial cells, retinal pericytes, kidney, glomerular tissue,glomerular podocytes, renal glomerular endothelial cells, mesangialcells, cytotrophoblasts, syncytiotrophoblast, human brain microvascularendothelial cells, human neural stem cells, astrocytes, neuroblastomacells, neural progenitor cells, placental endothelial cells, placentalfibroblasts, Hofbauer cells, amniotic epithelial cells, chorionic villicells, keratinocytes, dermal fibroblasts, dendritic cells, umbilicalvein endothelial cells, aortic endothelial cells, coronary arteryendothelial cells, saphenous vein endothelial cells, glial cells,primary spermatocytes, Sertoli cells, retinal bipolar cells, retinalganglion cells, optic nerve cells, and Vero cells. It is desirable todeliver the antiviral agent to such targets because they are the sitesof infection and replication. Targeted delivery could also preventunwanted effects on other tissues or organs. In an alternate embodiment,the method comprises administering the antiviral agent locally to thesubject's eye.

A method of reducing or preventing the replication of ZIKV in a hostcell is provided, the method comprising contacting the host cell with aneffective concentration any of the antiviral agents described above. Ina specific embodiment of the method the effective concentration is atleast about 10, 12, 15, or 20 μM. In a further specific embodiment ofthe method the effective concentration is about 10, 12, 15, or 20 μM, orany subrange thereof. The host cell may be situated in vivo or ex vivo,and may be any cell type known to be permissive to ZIKV, including anyof those listed above.

A method of controlling the spread of ZIKV in donated tissue isprovided, the method comprising exposing the donated tissue to aneffective amount of any embodiment of the antiviral agent disclosedabove. The donated tissue may be in the form of a donated organ. Theorgan or tissue may be exposed to the antiviral agent by perfusing theorgan or tissue with a solution containing the effective concentrationof the antiviral agent. In a specific embodiment of the method theeffective concentration is at least about 10, 12, 15, or 20 μM. In afurther specific embodiment of the method the effective concentration isabout 10, 12, 15, or 20 μM, or any subrange thereof. The antiviral agentmay be part of an organ preservation composition, such as University ofWisconsin cold storage solution (available from Bridge to Life Ltd.,Columbia, S.C.) or any other organ preservation solution known in theart. Another aspect of the disclosed work is a treated donated organ ortissue, comprising an organ preservation composition that includes aneffective amount of any of the antiviral agents listed above.

E. Working Example 1

The use of PMO based technology targeting the nucleotide translationinitiation complex site of ZIKV for antiviral development was explored.

Human glomerular podocytes were obtained from Dr. Moin A. Saleem [14]and were cultured as described in [15]. All cells were trypsinized andplated on uncoated 4.2 cm²/well glass chamber slides at density 2.5×10⁵cells per well or in 6 well dishes at a concentration 3.5×10⁵ per well.

A lyophilized, modified PMO was dissolved in sterile water to a finalconcentration of 0.5 mM. The PMO was a 25-mer having the sequence 5′-CATGAC CAG AAA CTC TCG TTT CCA A-3′ (SEQ ID NO: 4). The PMO was modified bythe addition of an octa-guanidine dendrimer of the following structure:

This modified PMO was dubbed DWK-M1.

A 30 μL aliquot was added to podocytes cultured in fresh 1.5 mL RPMImedia supplemented with 2% FCS and insulin-transferrin-selenium (ITS)per well of 6-well dishes. The final concentration of the modified PMOculture medium was 10 μM. After 24 hours of incubation, podocytes wererinsed with RPMI supplemented with 10% FCS and ITS and either mockinfected or infected with ZIKV and cultured for the indicated time.

The ZIKV strain PRVABC59 was used, originally isolated from a humanserum specimen from Puerto Rico in December 2015, nucleotide (GenBank):KU501215 ZIKV strain PRVABC59 [1-3]. The virus was cultivated in Verocells (Cercopithecus aethiops, kidney cell line) and infectioussupernatant was filtered using a 0.22 μm filter and the serum contentadjusted to 15%. Stock viral titers were done by florescent focus assays(FFA) on Vero cells using the 4G-2 Flavivirus group antigen monoclonalantibodies from Millipore (Temecula, Calif., USA) (“4G-2 antibody”) andwas adjusted to about 1×10⁴ particles/5 μL of infectious culturesupernatant.

Total RNA was extracted from ZIKV infected podocytes, along with therespective mock infected cells, or podocytes pretreated with control orthe modified PMOs and infected with ZIKV using a Qiagen RNeasy Mini Kit(Qiagen, Valencia, Calif., USA). Messenger RNA in 0.5 μg of each samplewas primed using random hexamers and reverse transcribed with a highcapacity cDNA reverse transcription kit (Applied Biosystems, FosterCity, Calif., USA). Real-time quantitative PCR was performed oniCycler96 using iQ Sybr Green Supermix (Bio-Rad). Samples were analyzedin triplicate and normalized to GAPDH RNA. Reaction mixture contained250 nM of each primer and 200 to 400 ng of template cDNA in a finalvolume of 20 μL. The primers specific for ZIKV were as follows: forward5′ AGG ATC ATA GGT GAT GAA GAA AAG T 3′ (SEQ ID NO: 6) and reverse 5′CCT GAC AAC ACT AAG ATT GGT GC 3′ (SEQ ID NO: 7) [4]. GAPDH primers usedfor qRT-PCR were as follows: forward: 5′-GAA GGT GAA GGT CGG AGT-3′ (SEQID NO: 8) and reverse: 5′-GAA GAT GGT GAT GGG ATT TC-3′ (SEQ ID NO: 9).

Immunofluorescent staining was performed. Briefly, chamber slidecultures containing mock infected human podocytes, podocytes infectedwith ZIKV and podocytes infected ZIKV after 24 hours pre-treatment withthe modified PMO. Cells were washed twice with PBS pH 7.4, air dried,and fixed in absolute methanol for 20 min at −20° C. Cells were airdried for 10 min, hydrated in Tris buffered saline (pH 7.6) for 10 min,and incubated for 1 h with 4G-2 antibody at a dilution 1:100 in PBS pH7.4 [5].

For the western blot analysis, cell extracts were prepared using RIPAlysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 2 mMethylenediaminetetraacetic acid (EDTA) pH 8.0, 1% NP40, 0.5% sodiumdeoxycholate, 0.1% sodium dodecyl sulfate (SDS), and proteinaseinhibitor (Complete Ultra, Roche)). Lysates were incubated on ice for 30minutes and then clarified by centrifugation. Total protein was measuredby micro BCA protein assay kit (ThermoFisher Scientific). 30 μg ofprotein lysates from paired, mock and ZIKV and PMO controls with andwithout ZIKV infection were separated by 10% SDS-PAGE gels, transferredto nitrocellulose membranes (Bio-Rad), blocked with 5% milk in 0.1% TBST(0.1% Tween 20, 20 mM Tris, 150 mM NaCl) and incubated at 4° C.overnight with 4G-2 antibody at 1:250 dilution. Synaptopodin antibody(Santa Cruz Biotechnology) was used at 1:250 dilution and GAPDH antibody(Santa Cruz Biotechnology) at 1:3000 dilution. Membranes were washedfive times in 0.1% TBST and incubated for one hour with correspondingsecondary antibody conjugated with HRP (ThermoFisher Scientific) at adilution of 1:50,000. Immunoreactive bands were detected withWesternBright ECL (Advansta) following exposure to X-ray film.

Experiments presented in this example were performed in triplicate. Tocompare the mean values between two groups, the unpaired t-test wasused. Statistical significance was defined as P<0.05. Data are presentedas means±SD. qRT-PCR experiments were replicated three times andnormalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Advantageously, the modified PMO was found to inhibit ZIKV transcriptionin infected human glomerular podocytes. Without being bound by anyparticularly theory, it is believed that ZIKV enters a permissive cellvia receptor mediated endocytosis. Acidification of the endosome resultsin breakdown of the viral nucleocapsid and release of the positive,sense genomic RNA. The modified PMO efficiently binds to ZIKV genomicRNA to block translation of the ZIKV polyprotein precursor. The modifiedPMO was found to inhibit ZIKV transcription in infected human glomerularpodocytes that are highly permissive for ZIKV infection. As qRT-PCRshowed, podocytes pretreated for 24 hours prior to ZIKV infection with10 μM of the modified PMO can reduce ZIKV RNA expression by 1438-fold(99.9% reduction) after 72 hours as compared to mock and ZIKV infectedcontrols (FIG. 1A).

The test was repeated to include mock infected podocytes, podocytesexposed to a control PMO, podocytes exposed to the control PMO+ZIKVinfected, podocytes exposed to the modified PMO alone, podocytesinfected with wildtype ZIKV, and podocytes exposed to the modifiedPMO+ZIKV infected (FIG. 1B). Results showed that mock infectedpodocytes, podocytes exposed to the control PMO or the modified PMOalone showed no detectable (ND) levels of ZIKV RNA expression afteramplification (FIG. 1B). Podocytes exposed the control PMO and infectedwith ZIKV showed increased levels ZIKV RNA compared to mock infectedcontrols. Podocytes exposed to wildtype ZIKV showed increased levels ofZIKV RNA expression but podocytes exposed to the modified PMO andinfected with wildtype ZIKV showed 94% decrease in ZIKV RNA expressioncompared to podocytes infected with wildtype ZIKV (FIG. 1B).

The modified PMO was found to inhibit ZIKV replication and proteinsynthesis in podocytes to undetectable levels. To determine if themodified PMO inhibition ZIKV transcription in infected human glomerularpodocytes would result in a decrease in ZIKV protein expression, ZIKVtotal protein expression after the modified PMO treatment of infectedpodocytes by immunofluorescent staining and immunoblot analysis wasexamined (FIGS. 2 and 3). The 4G-2 antibody did not stain mock infectedpodocytes, while podocytes infected with wildtype ZIKV for 72 hoursshowed characteristic perinuclear staining with the 4G-2 antibody (FIG.2). Podocytes pre-treated with the modified PMO and infected withwildtype ZIKV for 72 hours showed no specific expression of ZIKVproteins after infection as shown by negative staining with the 4G-2antibody and by comparison to mock infected cells (FIG. 2). In addition,ZIKV total E-protein expression after treatment with the modified PMOfor 72 hours was subjected to immunoblot analysis (FIG. 3). In theimmunoblot analysis, it was observed that ZIKV E-protein expression inboth podocytes infected with wildtype ZIKV and podocytes exposed to thePMO control (Co DWK)+ZIKV. Higher levels of ZIKV E-protein was observedin podocytes infected with wildtype ZIKV compared to podocytes exposedto the PMO control (Co DWK)+ZIKV (FIG. 3). However, podocytes pretreatedwith the modified PMO and infected with wildtype ZIKV showed nodetectable levels of ZIKV E-protein (FIG. 3). Moreover, the expressionof the podocyte marker Synaptopodin was not effected by ZIKV infectionor podocyte exposure to the control PMO or the modified PMO (FIG. 3).

The modified PMO was found to inhibit ZIKV induced inflammation inpodocytes. Mock infected podocytes, podocytes exposed to the controlPMO, podocytes exposed to the control PMO+ZIKV, podocytes exposed to themodified PMO alone, podocytes infected with wildtype ZIKV, and podocytesexposed to the modified PMO+ZIKV by qRT-PCR for ZIKV induction ofRANTES, MIP-1alpha, TNF-alpha, and INF-b were examined (FIGS. 4A-4D).Results showed increased levels of RANTES (FIG. 4A), MIP-1 alpha (FIG.4B), TNF alpha (FIG. 4C) and INF-b (FIG. 4D) in ZIKV infected podocytescompared to control cells that were not exposed to ZIKV (FIGS. 4A-4D).An upregulation of RANTES expression was observed 72 hours after ZIKVinfection in both podocytes exposed to the control PMO+ZIKV and inpodocytes infected with wildtype ZIKV (FIG. 4A). However, a suppressionof RANTES transcription was observed in podocytes pretreated with themodified PMO prior to ZIKV infection as compared to levels detected inpodocytes exposed to wildtype ZIKV (FIG. 4A). However, no induction ofRANTES expression was detected in mock podocytes or podocytes exposed tothe modified PMO alone (FIG. 4A). Lower levels of RANTES transcriptionwere observed in podocytes exposed to the control PMO+ZIKV compared topodocytes infected with wildtype ZIKV only (FIG. 4A). Upregulation wasdetected of MIP-1 alpha, TNF-alpha and IFN-b mRNA expression 72 hoursafter ZIKV infection in both podocytes exposed to the control PMO+ZIKVand in podocytes infected with wildtype ZIKV (FIGS. 4B, 4C, and 4D).There was suppression of MIP-1 alpha, TNF-alpha, and IFN-b transcriptionin podocytes exposed to the modified PMO prior to ZIKV infectioncompared to podocytes exposed to wildtype ZIKV (FIGS. 4B, 4C, and 4D).However, no significant induction of MIP-1 alpha, TNF-alpha, and IFN-bexpression was detected in mock podocytes or podocytes exposed to themodified PMO alone (FIGS. 4B, 4C, and 4D). Lower levels were observed ofMIP-1 alpha, TNF-alpha, and IFN-b transcription in podocytes exposed tothe control PMO+ZIKV compared to podocytes infected with wildtype ZIKVonly (FIGS. 4B, 4C, and 4D).

In this example, the effectiveness of the ZIKV specific PMO (“themodified morpholino” or “DWK-M1”) was surprisingly found to suppressactive transcription of ZIKV in vitro by 1438-fold, or 99.9%. Themodified PMO was shown to reduce ZIKV total E-protein expression toundetectable levels. In addition, it was shown that the modified PMO hasno effect on the steady state expression levels of the podocyte specificbiomarker synaptopodin. Furthermore, it was shown that the modified PMOsuppresses ZIKV induced RANTES, MIP-1 alpha, TNF-alpha, and INF-b tolevels observed in mock infected control cells.

REFERENCES

-   1. Lanciotti R S, Lambert A J, Holodniyet M, et al. 2016. Phylogeny    of Zika Virus in Western Hemisphere, 2015. Emerg Infect Dis 2016; 5:    933-35.-   2. Thomas D L, Sharp T M, Torres J, et al. Local Transmission of    Zika Virus—Puerto Rico, Nov. 23, 2015-Jan. 28, 2016. MMWR Morb    Mortal Wkly Rep 2016; 6:154-58.-   3. Dirlikov E, Ryff K R, Torres-Aponte J, et al. 2016. Update:    Ongoing Zika Virus Transmission—Puerto Rico, Nov. 1, 2015-Apr.    14, 2016. MMWR Morb Mortal Wkly Rep 2016; 17:451-55.-   4. Xu M Y, Liu S Q, Deng C L, et al. Detection of Zika virus by SYBR    green one-step real-time RT-PCR. J Virol Methods 2016; 236:93-7.-   5. Wilkerson I, Laban J, Mitchell J M, et al. Retinal pericytes and    cytomegalovirus infectivity: implications for HCMV-induced    retinopathy and congenital ocular disease. J Neuroinflammation 2015;    12: 2.

F. Working Example 2

In Working Example 2, the use of a morpholino oligonucleotide targetedto the 5′ untranslated region (5′-UTR) of the ZIKV RNA to prevent ZIKVreplication was explored. Vivo-morpholino oligonucleotide DWK-1 was usedat 10 μM concentration, and inhibition of ZIKV replication in humanglomerular podocytes treated with DWK-1 was analyzed by qRT-PCR,reduction in ZIKV genome copy number, western blot analysis,immunofluorescence and proinflammatory cytokine gene expression in ZIKVinfected podocytes pretreated with DWK-1. An approximately 95% reductionin ZIKV transcription in podocytes pretreated with DWK-1 followed by 72h exposure to ZIKV when compared to controls was shown.Immunofluorescence assay and immunoblot analysis showed highly reducedlevels of ZIKV E protein expressed in infected podocytes pretreated withDWK-1. Also observed was a robust suppression of proinflammatory geneexpression, IFN-β (interferon β) RANTES (regulated on activation, normalT cell expressed and secreted), MIP-1α (macrophage inflammatoryprotein-1α), TNF-α (tumor necrosis factor-α) and IL1-α (interleukin 1-α)in ZIKV-infected podocytes pretreated with DWK-1. Thus, Working Example2 found that Morpholino DWK-1 targeting the ZIKV 5′-UTR effectivelyinhibited ZIKV replication and suppressed ZIKV-induced proinflammatorygene expression. Working Example 2 is described in further detail,below, with sections and subsections used for organizational purposes.

Materials and Methods

Morpholino Oligomers

The ZIKV-targeted morpholino oligomer DWK-1 was designed to becomplementary to the 25-mer nucleotide sequence within the ZIKV 5′untranslated region (5′-UTR) (bolded in brackets) that includes thefirst ATG translation start codon (bolded, underlined) of the Zika virusstrain PRVABC59 (GenBank mRNA transcript KU501215.1,PRVABC59/Puerto-Rico/2015): 5′-GTA TCA ACA GGT TTT ATT TTG GAT [TTG GAAACG AGA GTT TCT GGT CATG]AAA AAC CCA AAA AAG AAA TCC G-3′ (SEQ ID NO:10). The 5′-UTR of the ZIKV PRVABC59 RNA sequence targeted by DWK-1 ishighly conserved among ZIKV strains. The sequence of DWK-1 complementaryto the 25-mer of ZIKV5′-UTR is as follows: 5′-CAT GAC CAG AAA CTC TCGTTT CCA A-3′ (SEQ ID NO: 3). The control oligo used in this Example wasa standard control oligo that targets a human beta-globin intronmutation that causes beta-thalassemia. This oligo, designated as CoDWK-1, causes little change in phenotype in any known test system excepthuman beta-thalassemic hematopoietic cells and is appropriate negativecontrol for custom vivo-morpholino oligos (Moulton, 2017). The sequenceof Co DWK-1 is as follows: 5′-CCT CTT ACC TCA GTT ACA ATT TAT A-3′ (SEQID NO: 11). Morpholino oligonucleotides used (vivo-morpholinos) wereconjugated to a delivery moiety consisting of an eight-brancheddendrimer carrying a guanidinium moiety at each branch tip (see FIG. 5)for efficient delivery of morpholino to the cytosol and nuclearcompartments of the cell. The vivo-morpholinos DWK-1 and Co DWK-1 weresynthesized by Gene Tools, LLC. The rationale for using 25-mers which isthe longest commercially available morpholino is that they arerecommended for most applications. This is because efficacies increasesubstantially with increasing length and because long oligos best assureaccess to a single-stranded region in the target RNA, as is required fornucleation of pairing by the oligo. This length versus activity studywas carried out by Gene Tools with morpholino oligos and 25 mers werefound to be the optimal length for sequence specific knockdown of genesin mammalian cells.

Cells

Immortalized human glomerular podocytes AB8/13 were obtained from MoinA. Saleem (Saleem et al., 2002) and were cultured as described (Khatuaet al., 2010). Cells were trypsinized and plated in 6 well dishes at aconcentration 3.5×10⁵ per well. The cells were cultured in RPMI mediasupplemented with 10% FCS and insulin-transferrin-selenium (ITS;ThermoFisher Scientific).

Morpholino Pretreatment

Lyophilized morpholino oligos DWK-1 and Co-DWK-1 were dissolved insterile water to a final concentration of 0.5 mM. A 30 μL aliquot wasadded to podocytes cultured in fresh 1.5 mL RPMI media supplemented with10% FCS and ITS per well of 6-well dishes. The final concentration ofDWK-1 and Co DWK-1 in culture medium was 10 μM. After 24 h incubation,podocytes were rinsed with culture medium and either mock infected orinfected with ZIKV and cultured for the indicated time in the absence ofmorpholinos.

ZIKV Preparation and Titration

The Zika virus strain PRVABC59 used in this study was originallyisolated from a human serum specimen from Puerto Rico in December 2015,nucleotide (GenBank): KU501215 ZIKV strain PRVABC59, complete genome(Lanciotti et al., 2015; Thomas et al., 2016; Dirlikov et al., 2016;Lancontti et al., 2008). The virus was cultivated in Vero cells andinfectious supernatant was filtered using a 0.22 μm filter and the serumcontent adjusted to 15%. Stock viral titers were determined aspreviously described (Alcendor, 2017). All experiments were carried outunder biosafety level-2 containment as recommended. Use of ZIKV wasapproved by the Meharry Medical College Institutional Review Board andthe Institutional Biosafety Committee.

ZIKV RNA Analysis

Total cellular RNA was isolated from the cells using Quick RNA MiniPrepkit (Zymo Research) and 500 ng RNA was reverse transcribed into cDNAusing iScript cDNA synthesis kit (Bio-Rad). Real-time PCR was performedon CFX96 PCR machine (Bio-Rad) using SYBR Green PCR master mix(Bio-Rad), ZIKA specific primers (forward primer 5′-CCG CTG CCC AAC ACAAG-3′ (SEQ ID NO: 12) and reverse primer 5′-CCA CTA ACG TTC TTT TGC AGACAT-3′ (SEQ ID NO: 13)) and GAPDH specific primers (forward 5′-GAA GGTGAA GGT CGG AGT-3′ (SEQ ID NO: 8) and reverse 5′-GAA GAT GGT GAT GGG ATTTC-3′ (SEQ ID NO: 9)). The following amplification conditions were used:95° C. for 3 min for initial denaturation and 40 cycles of 95° C. for 10s and 60° C. for 45 s. Samples were analyzed in triplicate and ZIKV RNAexpression was normalized to GAPDH mRNA levels. Data are presented asmean±SD. A standard curve was generated by using the 10-fold serialdilutions of a synthetic ZIKV RNA (ATCC VR-3252SD) with known ZIKVgenome copies (provided as 1.2×10⁶ copies/μL). Absolute quantificationof ZIKV genome copy numbers was carried out in triplicate by comparingeach sample's threshold cycle (CT) value with a ZIKV RNA standard curve.

qRT-PCR Analysis of the Proinflammatory Cytokine Gene Expression

Total cellular RNA was isolated, processed, and analyzed as describedabove. The primers used to analyze cytokine gene expression are asfollows: IFN-β: forward 5′-CTT GGA TTC CTA CAA AGA AGC AGC-3′ (SEQ IDNO: 14), reverse 5′-TCC TCC TTC TGG AAC TGCT GCA-3′ (SEQ ID NO: 15);RANTES: forward 5′-CCT GCT GCT TTG CCT ACA TTG C-3′ (SEQ ID NO: 16),reverse 5′-ACA CAC TTG GCG GTT CTT TCG G-3′ (SEQ ID NO: 17); MIP-1α:forward 5′-ACT TTG AGA CGA GCA GCC AGT G-3′ (SEQ ID NO: 18), reverse5′-TTT CTG GAC CCA CTC CTC ACT G-3′ (SEQ ID NO: 19); TNF-α: forward5′-CTC TTC TGC CTG CTG CAC TTT G-3′ (SEQ ID NO: 20), reverse 5′-ATG GGCTAC AGG CTT GTC ACT C-3′ (SEQ ID NO: 21); IL-1α: forward 5′-TGT ATG TGACTG CCC AAG ATG AAG-3′ (SEQ ID NO: 22), reverse 5′-AGA GGA GGT TGG TCTCAC TAC C-3′ (SEQ ID NO: 23); IL-6: forward 5′-AGA CAG CCA CTC ACC TCTTCA G-3′ (SEQ ID NO: 24), reverse 5′-TTC TGC CAG TGC CTC TTT GCT G-3′(SEQ ID NO: 25).

Samples were analyzed in triplicate and cytokine gene expression wasnormalized to GAPDH mRNA levels.

Immunofluorescence

Immunofluorescent staining was performed as previously described(Alcendor, 2017). Briefly, chamber slide cultures containing mockinfected human podocytes, podocytes infected with ZIKV, and podocytesinfected ZIKV after 24 hours pre-treatment with DWK-1. Cells were washedtwice with PBS pH 7.4, air dried, and fixed in absolute methanol for 20min at −20° C. Cells were air dried for 10 min, hydrated in Trisbuffered saline (pH 7.6) for 10 min, and incubated for 1 h with the 4G-2Flavivirus group antigen monoclonal antibodies from Millipore (Temecula,Calif., USA) at a dilution 1:100 in PBS pH 7.4. (Wilkerson et al.,2015).

Western Blot Analysis

Cell extracts were prepared using RIPA lysis buffer [50 mM Tris pH 7.5,150 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA) pH 8.0, 1%NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), andproteinase inhibitor (Complete Ultra, Roche). Lysates were incubated onice for 30 min and then clarified by centrifugation. Total protein wasmeasured by micro BCA protein assay kit (ThermoFisher Scientific).Protein lysates (30 μg) were separated by 10% SDS-PAGE, transferred tonitrocellulose membranes (Bio-Rad), blocked with 5% milk in 0.1% TBST(0.1% Tween 20, 20 mM Tris, 150 mM NaCl) and incubated at 4° C.overnight with 4G-2 Flavivirus group antigen monoclonal antibody(Millipore, Temecula, Calif., USA) at 1:250 dilution. Synaptopodinantibody (Santa Cruz Biotechnology) was used at 1:250 dilution and GAPDHantibody (Santa Cruz Biotechnology) at 1:3000 dilution. Membranes werewashed five times in 0.1% TBST and incubated for one hour withcorresponding secondary antibody conjugated with HRP (ThermoFisherScientific) at a dilution of 1:50,000. Immunoreactive bands weredetected with WesternBright ECL (Advansta) following exposure to X-rayfilm.

Statistical Analysis

Experiments presented in this study were performed independently threetimes under similar conditions. Data are presented as means withstandard deviations. Unpaired t-test was used to compare the mean valuesbetween groups. Differences were considered statistically significant atP<0.05.

Results

DWK-1 Inhibits Accumulation of Intracellular ZIKV RNA in aDose-Dependent Manner

To determine an effective concentration of vivo-morpholino DWK-1 (FIG.5) that inhibits ZIKV replication in human podocytes, the cells werepretreated for 24 h with various concentrations of DWK-1 and Co DWK-1ranging from 1 to 10 μM, rinsed and mock infected or infected with ZIKV(PRVABC59) at a multiplicity of infection (MOI) of 0.1 in the absence ofmorpholinos. Seventy two hours after infection, the cells were collectedand intracellular ZIKV RNA accumulation was determined by qRT-PCR (FIG.6). Results show that DWK-1 reduced intracellular ZIKV RNA accumulationin a dose-dependent concentration with about 50% inhibition of ZIKV RNAaccumulation at 1.0-1.5 μM and >95% inhibition at 10 μM. In contrast,Co-DWK-1 shows only a small inhibition (9±5%) at 10 μM. Since 10 μMconcentration of DWK-1 was of low toxicity to the cells, it was used inall Working Example 2 experiments.

DWK-1 Reduces Expression of Intracellular ZIKV RNA in Podocytes

To validate further the antiviral activity of DWK-1, ZIKV RNA copynumber was measured in infected podocytes or podocytes pretreated withDWK-1 or Co DWK-1 and subsequently infected with ZIKV. First, a standardcurve was generated by using 10-fold dilutions of synthetic ZIKV RNA(ATCC VR-3252SD) (FIG. 8A). The standard curve covered a linear rangefrom 10⁶ to 10 copies of ZIKV RNA with a slope=−3.923 and R²=0.997,indicating a good sensitivity of the SYBR Green qRT-PCR assay (FIG. 8B).Total cellular RNA was isolated from podocytes treated as indicated inFIG. 8C and analyzed by qRT-PCR for the expression of ZIKV and GAPDHtranscripts. Results demonstrate 95% reduction of ZIKV RNA expression inpodocytes pretreated with DWK-1 and infected for 48 h with ZIKV, ascompared to infected podocytes pretreated with Co DWK-1. These resultscorrelated with an about 94% reduction of ZIKV RNA copy number (FIG. 8D)as quantitated from a standard curve (FIG. 8B) generated using syntheticZIKV RNA.

DWK-1 Strongly Reduces Expression of ZIKV E Protein in InfectedPodocytes

To determine if DWK-1 inhibition of ZIKV transcription in infected humanglomerular podocytes results in a decrease in ZIKV protein expression,expression of ZIKV E protein in podocytes pretreated with DWK-1 wasexamined. Immunofluorescent staining showed that E protein-specific 4G-2antibody does not stain mock infected podocytes, while podocytesinfected with ZIKV for 72 h showed characteristic perinuclear stainingwith the 4G-2 antibody (FIGS. 9A-9D). In contrast, podocytes pretreatedwith DWK-1 and infected with ZIKV for 72 h showed only a minimal, ifany, expression of ZIKV E protein as compared to mock and isotypecontrols (FIG. 9A-9D). Similarly, expression of ZIKV E protein ininfected podocytes after pretreatment with DWK-1 (ZIKV+DWK-1) wasstrongly reduced (>98%) by immunoblot analysis (FIG. 10). No E proteinexpression was observed in uninfected (Mock, Co DWK-1, DWK-1) podocytes.Expression of the podocyte biomarker Synaptopodin was demonstrated notto be significantly affected by ZIKV infection or podocyte exposure toDWK-1 or Co DWK-1 (FIG. 10).

DWK-1 Inhibits ZIKV-Induced Proinflammatory Gene Expression in Podocytes

ZIKV virus infection leads to the induction of proinflammatorycytokines. It was examined whether DWK-1 pretreatment affects expressionof proinflammatory cytokine genes in ZIKV infected podocytes (FIGS.11A-11D). Surprisingly, ZIKV induced a robust 4,023-fold increase inIFN-β gene expression and 3,330-fold increase in podocytes pretreatedwith Co DWK-1 (FIG. 11A), when compared to mock infected cells.Importantly, pretreatment with DWK-1 prior to ZIKV infection resulted inover a 16-fold suppression of IFN-β transcriptional expression, ascompared to cells pretreated with Co DWK-1 (FIG. 11A). Similarly, astrong upregulation of RANTES transcriptional expression at 72 h afterinfection with ZIKV (58.8-fold increase) and in cells pretreated with CoDWK-1 and infected with ZIKV (47.2-fold increase) (FIG. 11B) wasobserved. Pretreatment of podocytes with DWK-1 prior to ZIKV infectionresulted in >9-fold reduction in RANTES gene expression, when comparedto levels detected in infected podocytes pretreated with Co DWK-1 (FIG.11B). No significant changes in the RANTES transcriptional expressionwere observed in mock podocytes or podocytes exposed to the DWK-1 or CoDWK-1 alone (FIG. 11B). Although the expression of MIP-1 α, TNF-α andIL-1α was not so potently induced by ZIKV in podocytes (˜2 to 4-foldupregulation) when compared to IFNβ or RANTES, pretreatment with DWK-1prior to ZIKV infection reduced expression of these genes to levelsdetected in mock infected podocytes (FIGS. 11C-11E). No significantchanges in IL-6 transcriptional expression were detected in podocytesexposed to DWK-1 prior to ZIKV infection, when compared to infectedpodocytes preexposed to Co DWK-1 (FIG. 11F).

Discussion

In this Working Example, the effectiveness of the ZIKV targetedmorpholino DWK-1 was demonstrated to suppress active transcription ofZIKV in vitro by approximately 95% and to reduce ZIKV E proteinexpression to undetectable levels. In addition, it was shown that DWK-1has no effect on the steady state expression levels of the podocytespecific biomarker synaptopodin. It was also shown that DWK-1 potentlyreduced expression of IFN-β, RANTES, MIP-1α and TNF-α in ZIKV infectedcells, as compared to infected cells pretreated with Co DWK-1.

Advantageously, the antiviral agents described herein have the potentialto be highly useful as prophylaxis or treatment for immunosuppressedSOTp receiving allografts from ZIKV infected donors as well as anant-infective for protecting a blood supply tainted with ZIKV especiallyin ZIKV endemic regions where ZIKV screening of blood is unavailable.

These antivirals agents, which inhibit active replication of ZIKV, wouldbe beneficial for these patients and could potentially suppress sporadicoutbreaks of ZIKV infection in the general population. Such an antiviralagent that is stable at room temperature could be highly useful in aridconditions without refrigeration. This advantage could also enabledevelopment of a carry-on intervention to prevent ZIKV infection formilitary personnel and humanitarian workers traveling to ZIKV endemicregions.

Conclusions

The vivo-morpholino is composed of a morpholino oligo with a uniquecovalently linked delivery moiety, which is comprised of anocta-guanidine dendrimer. The active component, namely the arginine richdelivery peptides of the guanidinium group facilitates delivery of themodified morpholino into the cytosol. In this Working Example, amorpholino-based antiviral was shown to target ZIKV 5′-UTR, be oflow-toxicity, be stable at room temperature, and is capable ofpenetrating target cells.

REFERENCES

-   Alcendor D J, Zika Virus Infection of the Human Glomerular Cells:    Implications for Viral Reservoirs and Renal Pathogenesis. J Infect    Dis 2017 jix171. doi: 10.1093/infdis/jix171-   Dirlikov E, Ryff K R, Torres-Aponte J, et al. 2016. Update: Ongoing    Zika Virus Transmission—Puerto Rico, Nov. 1, 2015-Apr. 14, 2016.    MMWR Morb Mortal Wkly Rep 2016; 17:451-55.-   Khatua A K, Taylor H E, Hildreth J E, et al. Non-productive HIV-1    infection of human glomerular and urinary podocytes. Virology 2010;    1:119-27.-   Lanciotti, R. S., Kosoy, O. L., Laven, et al. Genetic and serologic    properties of Zika virus associated with an epidemic, Yap State,    Micronesia, 2007. Emerg Infect Dis 2008; 14: 1232-39.-   Lanciotti R S, Lambert A J, Holodniyet M, et al. 2016. Phylogeny of    Zika Virus in Western Hemisphere, 2015. Emerg Infect Dis 2016; 5:    933-35.-   Moulton J D. Using morpholinos to control gene expression. Curr.    Protoc. Nucleic Acid Chem. 2017; 68:4.30.1-4.30.29.-   Saleem M A, O'Hare M J, Reiser J, et al. A conditionally    immortalized human podocyte cell line demonstrating nephrin and    podocin expression. J Am Soc Nephrol 2002; 3:630-8.-   Thomas D L, Sharp T M, Torres J, et al. Local Transmission of Zika    Virus—Puerto Rico, Nov. 23, 2015-Jan. 28, 2016. MMWR Morb Mortal    Wkly Rep 2016; 6:154-58.

G. Working Example 3

An MPO was designed that targets a sequence in the sHP-3′SL region ofthe 3′UTR of ZIKV strains. This is a highly conserved region (FIGS. 12and 13). The MPO, designated DWK-2, targets the sequence 5′-GCT GGG AAAGAC CAG AGA CTC CAT G-3′ (SEQ ID NO: 4) (FIG. 13), and has the primarysequence 5′-CAT GGA GTC TCT GGT CTT TCC CAG C-3′ (SEQ ID NO: 5).

The inhibition of ZIKV-induced proinflammatory cytokine gene expressionby DWK-2 was measured. Podocytes were pretreated for 24 h with 10 μMDWK-2 or Co DWK-1 (control) and infected with ZIKV. Mock infected cellsand cells treated only with DWK-2 or Co DWK-2 were included as controls.Total RNA was isolated at 72 h p.i. and intracellular ZIKV RNA wasquantitated by qRT-PCR and normalized to GAPDH mRNA levels. Results inFIGS. 14A-14D show inhibitory effect of DWK-2 on the expression of ZIKVinduced (FIG. 14A) IL-6, (FIG. 14B) IL-1α, (FIG. 14C) INF-β, and (FIG.14D) RANTES genes. Values represent mean±SD of 3 independent samples.The expression of cytokine genes mRNA in mock infected cells was set as1.0.

The inhibition of accumulation of intracellular ZIKV RNA in infectedpodocytes was measured. Podocytes were pretreated for 24 h with 10 μMDWK-2 or Co DWK-2 (control) and infected with ZIKV. Mock infected andDWK-2 and Co DWK-2 pretreated cells were included as controls. Total RNAwas isolated at 72 h p.i. and intracellular ZIKV RNA expression wasdetermined by qRT-PCR and normalized to GAPDH mRNA levels. ZIKVinfections were performed in triplicate. Results are shown in FIG. 15A.Values represent mean±SD of 3 independent samples. ND, not detected.

The effect of DWK-2 on ZIKV RNA genome copy number in infected podocyteswas tested. Total cellular RNA isolated from mock, ZIKV infected cells,or cells pretreated for 24 h with 10 μM DWK-2 alone, or from DWK-2pretreated cells and infected with ZIKV for 48 h was analyzed by qRT-PCRfor the expression of ZIKV and GAPDH RNA. Results are shown in FIG. 15B.Relative expression of intracellular ZIKV RNA normalized to GAPDH RNA isreduced by 94.2%. Quantitation of ZIKV genome copy number in totalintracellular RNA shows a reduction in ZIKV copy number in infectedcells pretreated with DWK-2. Values represent mean±SD of 3 independentsamples. ND, not detected.

H. Working Example 4

The toxicity of DWK-1 in mice was determined. DWK-1 toxicity data shownin CD-1 mice was performed by Pacific Biolab, Hercules Calif. (FIG. 7).A summary of the data that includes animal grouping, dosing regimen andmortality 96 h after s.c. injection is shown. All animals survived thehighest dose of 30 mg/kg after temporary vasodilation and hypoactivityimmediately after the dose. All groups recovered from temporaryvasodilation and hypoactivity within 3 hours, with only a scruffyappearance and slight vasodilation. These data indicate that DWK-1 isnon-toxic in CD-1 mice and support testing DWK-1 in a murine model forZIKV infection.

I. Prophetic Example 5

DWK-1 dosing and toxicity in the murine model prior to ZIKV exposure.Multiple dosing of DWK-1 will be performed over a period 96 hours inCD-1 mice. The experiment will also be performed in pregnant female micewith the intention to examine ill effects of DWK-1 on the mother andpups. Multiple dosing (1 dose per day for 4 days) in 50 μl volumes willbe administered intraperitoneally (i.p.). Animals will be examined dailyfor ill effects and pups will be examine after birth for toxicity andevidence of pathology. ZIKV infection of the murine model. Animals:Utilizing a predetermined dosing regimen for DWK-1 from previoustoxicity studies an efficacy evaluation will be performed of DWK-1 inthe ZIKV infected murine model previously described (Miner J J, Sene A,Richner J M, Smith A M, Santeford A, Ban N, Weger-Lucarelli J, ManzellaF, Ruckert C, Govero J, Noguchi K K, Ebel G D, Diamond M S, Apte R S.Zika Virus Infection in Mice Causes Panuveitis with Shedding of Virus inTears. Cell Rep. 2016; 20; 16(12):3208-3218). Animal studies in the ZIKVmurine model will be performed as a fee for service with WashingtonUniversity at St. Louis. All protocols will be approved by theInstitutional Animal Care and Use Committee at the Washington UniversitySchool of Medicine. Wild type C57BL/6 mice (Jackson Laboratories) willbe treated with 2 mg of an anti-Ifnar1 blocking mouse MAb (MAR1-5A3) orisotype control mouse MAb (GIR-208) (Leinco Technologies). Virus: TheZIKV strain H/PF/2013 (French Polynesia) and the ZIKV PRVABC59 will beused in this study. ZIKV infections: Four to eight-week-old anti-Ifnar1mice will be inoculated with ZIKV by the subcutaneous (footpad) routewith 103 FFU in 50 μl of PBS and control animals will be given PBS only.Evaluation of ZIKV infected Mice: Mice will be examined daily forevidence of disease and pathology. Harvested organs will be examined forZIKV infection by qRT-PCR. Evaluation of Animals Post Treatment: Treatedand control mice infected with ZIKV will be examined for evidence ofprotection against ocular disease, systemic infection, and maternaltransmission. In addition, animals will be assessed for toxicity, offtarget effects, viral loads in tissue and body fluids by qRT-PCR.Histological examinations of tissue will be done by immunohistochemistry(IHC).

Analysis of ZIKV infectivity in ocular tissue. Ocular tissue includingthe complete orbits of both the left and right eye of control and ZIKVinfected mice with and without DWK-1 treatment will be processedseparately as fresh frozen tissue (FFT) that will be stored in liquidnitrogen as well as formalin fix and paraffin embedded (FFPE) tissue.FFPE tissue will be laced on Chemate slides and H&E stained to examinegross pathology. Infected and control specimens will be stained by IHCfor ZIKV infection using the 4G2 antibody. FFT will be analyzed for ZIKVRNA by qRT-PCR. Tears and lacrimal glands from infected and controlanimals will be examined for viral burden by qRT-PCR.

Analysis of ZIKV infectivity in pregnant mice treated with DWK-1 toprevent ZIKV induced ocular disease in pups. The vertical transmissionof ZIKV in humans and the development of ocular disease in infants iswell documented but the underlying mechanisms are poorly understood.Therapeutic modalities to prevent intrauterine transmission of ZIKV arecurrently not available. The ability of DWK-1 will be examined toprevent intrauterine transmission of ZIKV to pups and to prevent ZIKVassociated CNS disease. Pregnant mice at the same gestational time pointwill be infected with ZIKV followed by a repeated subcutaneous dose of20 mg/kg of DWK-1. This dose will be repeated daily for 5 days. Animalswill be allowed to give birth and mother and pups will be examined forevidence of toxicity and ZIKV induced CNS pathology. Clinical andtranslational goals. Findings from the proposed studies will be utilizedas a basis for evaluating DWK-1 in a macaque model with futureimplications for Phase I testing in humans.

J. Examplary Embodiments

Embodiment 1: An antiviral agent that restricts the replication of Zikavirus (ZIKV) in a cell, the agent comprising a phosphorodiamidatemorpholino oligomer (PMO) comprising an antisense sequence to a portionof a genome of a strain of ZIKV.

Embodiment 2: A pharmaceutical composition for the treatment orprevention of a disease mediated by the Zika virus (ZIKV), thecomposition comprising: the antiviral agent of embodiment 1 and apharmaceutically acceptable carrier.

Embodiment 3: The pharmaceutical composition of embodiment 2, whereinthe pharmaceutically acceptable carrier is selected from the groupconsisting of: a vehicle, an adjuvant, a surfactant, a suspending agent,an emulsifying agent, an inert filler, a diluent, an excipient, awetting agent, a binder, a lubricant, a buffering agent, adisintegrating agent, an accessory agent, a coloring agent, and aflavoring agent.

Embodiment 4: The pharmaceutical composition of any one of embodiments2-3, wherein the antiviral agent is present in a therapeuticallyeffective amount.

Embodiment 5: The pharmaceutical composition of any one of embodiments3-4, wherein the therapeutically effective amount is sufficient toprovide the agent at a concentration of at least about 10 μM at a siteof viral infection in a subject.

Embodiment 6: The pharmaceutical composition of any one of embodiments3-5, wherein the therapeutically effective amount is a non-toxic amount.

Embodiment 7: The pharmaceutical composition of any one of embodiments3-6, wherein the therapeutically effective amount is sufficient toprovide the agent at a concentration of below an LD50 for a subject.

Embodiment 8: The pharmaceutical composition of any one of embodiments3-7, wherein the therapeutically effective amount is sufficient toprovide the agent at a dosage/body mass concentration of up to an amountselected from: 0.05, 0.1, 0.15, 0.2, 0.3, 0.5, 1, 1.5, 2, 3, 5, 10, 15,20, 30 mg/kg, about any of the foregoing values, and a range between anyof the foregoing values.

Embodiment 9: The pharmaceutical composition of any one of embodiments2-8, wherein the pharmaceutical composition is formulated to deliver theantiviral agent to a subject's circulatory system, placenta, fetus, eye,kidney, brain, skin, or any combination of the foregoing.

Embodiment 10: A method of treatment or prevention of a disease mediatedby the Zika virus (ZIKV) in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of the pharmaceutical composition of any one of embodiments 2-9.

Embodiment 11: The composition or method of any one of embodiments 2-10,wherein the disease mediated by ZIKV is selected from the followinggroup: Zika fever, Guillain-Barré syndrome, a congenital defect,microcephaly, ocular disease, and Zika associated organ pathology.

Embodiment 12: A method of reducing or preventing the replication ofZika virus (ZIKV) in a host cell, the method comprising contacting thehost cell with an effective amount of the antiviral agent of embodiment1.

Embodiment 13: The method of embodiment 12, wherein the host cell isselected from the group consisting of: a retinal endothelial cell, aretinal microvascular endothelial cell, a retinal pigmented epithelialcell, a retinal pericyte, a kidney cell, a glomerular podocyte, a renalglomerular endothelial cell, mesangial cell, cytotrophoblasts,syncytiotrophoblast, human brain microvascular endothelial cells, humanneural stem cells, astrocytes, neuroblastoma cells, neural progenitorcells, placental endothelial cells, placental fibroblasts, Hofbauercells, amniotic epithelial cells, chorionic villi cells, keratinocytes,dermal fibroblasts, dendritic cells, umbilical vein endothelial cells,aortic endothelial cells, coronary artery endothelial cells, saphenousvein endothelial cells, glial cells, primary spermatocytes, Sertolicells, retinal bipolar cells, retinal ganglion cells, optic nerve cells,Vero cells, and combinations thereof.

Embodiment 14: A method of controlling the spread of ZIKV in a specimenof donated tissue or organ, the method comprising exposing the specimento an effective amount of the antiviral agent of embodiment 1.

Embodiment 15: The method of embodiment 14, wherein the donated organ isselected from the group consisting of: heart, intestine, kidney, liver,lung, and pancreas; or the donated tissue is selected from the groupconsisting of: bone, cartilage, cornea, dura matter, fascia, heartvalve, ligament, pericardium, skin, tendon, and vein.

Embodiment 16: The method of any one of embodiments 14-15, comprisingperfusing the specimen with the antiviral agent.

Embodiment 17: The method of any one of embodiments 14-16, wherein theeffective amount is at least about 10 μM.

Embodiment 18: The method of any one of embodiments 14-17, wherein theeffective amount is a nontoxic amount.

Embodiment 19: A treated specimen of donated tissue or organ that is theproduct of the process of any one of embodiments 14-18.

Embodiment 20: Any one of embodiments 1-19, wherein the portion of thegenome of the strain of ZIKV is a 5′ portion comprising the untranslatedregion and the capsid protein.

Embodiment 21: Any one of embodiments 1-20, wherein the antisensesequence has at least 80% identity with 5′-CAT GAC CAG AAA CTC TCG TTTCCA A-3′ (SEQ ID NO: 3).

Embodiment 22: Any one of embodiments 1-21, wherein the antisensesequence has at least a level of identity with 5′-CAT GAC CAG AAA CTCTCG TTT CCA A-3′ (SEQ ID NO: 3) selected from the group consisting of:85%, 90%, 95%, 99%, and 100%.

Embodiment 23: Any one of embodiments 1-22, wherein the antisensesequence hybridizes under physiological conditions with RNA containingthe sequence 5′-TTG GAA ACG AGA GTT TCT GGT CAT G-3′ (SEQ ID NO: 2).

Embodiment 24: Any one of embodiments 1-23, wherein the antisensesequence hybridizes under highly stringent conditions with RNAcontaining the sequence 5′-TTG GAA ACG AGA GTT TCT GGT CAT G-3′ (SEQ IDNO: 2).

Embodiment 25: Any one of embodiments 1-19, wherein the portion of thegenome of the strain of ZIKV is a 3′ portion comprising the untranslatedregion.

Embodiment 26: Any one of embodiments 1-19, wherein the portion of thegenome of the strain of ZIKV is a structure in the 3′ portion comprisingthe untranslated region selected from the group consisting of: astem-and-loop structure, and a short hairpin structure.

Embodiment 27: Any one of embodiments 1-19, wherein the portion of thegenome of the strain of ZIKV is a structure in the 3′ portion comprisingthe untranslated region selected from the group consisting of: SL I, SLII, SL I, sHP, and the terminal 3′ end stem-and-loop structure.

Embodiment 28: Any one of embodiments 1-19 and 25-27, wherein theantisense sequence has at least 80% identity with 5′-CAT GGA GTC TCT GGTCTT TCC CAG C-3′ (SEQ ID NO: 5).

Embodiment 29: Any one of embodiments 1-19 and 25-28, wherein theantisense sequence has at least a level of identity with 5′-CAT GGA GTCTCT GGT CTT TCC CAG C-3′ (SEQ ID NO: 5) selected from the groupconsisting of: 85%, 90%, 95%, 99%, and 100%.

Embodiment 30: Any one of embodiments 1-19 and 25-29, wherein theantisense sequence hybridizes under physiological conditions with RNAcontaining the sequence 5′-GCT GGG AAA GAC CAG AGA CTC CAT G-3′ (SEQ IDNO: 4).

Embodiment 31: Any one of embodiments 1-19 and 25-30, wherein theantisense sequence hybridizes under highly stringent conditions with RNAcontaining the sequence 5′-GCT GGG AAA GAC CAG AGA CTC CAT G-3′ (SEQ IDNO: 4).

Embodiment 32: Any of embodiments 1-31, wherein the agent comprises amoiety for intracellular delivery.

Embodiment 33: Any of embodiments 1-32, wherein the agent comprises anocta-guanidine dendrimer delivery moiety.

Embodiment 34: Any of embodiments 1-33, wherein the agent comprises anocta-guanidine dendrimer of the following structure:

Embodiment 35: A use of the agent of any of embodiments 1 and 20-34 forthe manufacture of a medicament for the treatment or prevention of adisease mediated by the Zika virus (ZIKV).

Embodiment 36: The use of embodiment 35, wherein the disease mediated byZIKV is selected from the following group: Zika fever, Guillain-Barrésyndrome, a congenital defect, microcephaly, ocular disease, and Zikaassociated organ pathology.

Embodiment 37: A use of the agent of any of embodiments 1 and 20-34 forthe manufacture of a composition for controlling the spread of ZIKV in aspecimen of donated tissue or organ.

K. Conclusions

It is to be understood that any given elements of the disclosedembodiments of the invention may be embodied in a single structure, asingle step, a single substance, or the like. Similarly, a given elementof the disclosed embodiment may be embodied in multiple structures,steps, substances, or the like.

The foregoing description illustrates and describes the processes,machines, manufactures, compositions of matter, and other teachings ofthe present disclosure. Additionally, the disclosure shows and describesonly certain embodiments of the processes, machines, manufactures,compositions of matter, and other teachings disclosed, but, as mentionedabove, it is to be understood that the teachings of the presentdisclosure are capable of use in various other combinations,modifications, and environments and is capable of changes ormodifications within the scope of the teachings as expressed herein,commensurate with the skill and/or knowledge of a person having ordinaryskill in the relevant art. The embodiments described hereinabove arefurther intended to explain certain best modes known of practicing theprocesses, machines, manufactures, compositions of matter, and otherteachings of the present disclosure and to enable others skilled in theart to utilize the teachings of the present disclosure in such, orother, embodiments and with the various modifications required by theparticular applications or uses. Accordingly, the processes, machines,manufactures, compositions of matter, and other teachings of the presentdisclosure are not intended to limit the exact embodiments and examplesdisclosed herein. Any section headings herein are provided only forconsistency with the suggestions of 37 C.F.R. § 1.77 or otherwise toprovide organizational queues. These headings shall not limit orcharacterize the invention(s) set forth herein.

We claim:
 1. An antiviral agent that restricts the replication of Zikavirus (ZIKV) in a cell, the agent comprising a phosphorodiamidatemorpholino oligomer (PMO) comprising a sequence of 5′-CAT GAC CAG AAACTC TCG TTT CCA A-3′ (SEO ID NO: 3) or 5′-CAT GGA GTC TCT GGT CTT TCCCAG C-3′ (SEQ ID NO: 5).
 2. A pharmaceutical composition for thetreatment or prevention of a disease mediated by the Zika virus (ZIKV),the composition comprising: the antiviral agent of claim 1 and apharmaceutically acceptable carrier.
 3. The pharmaceutical compositionof claim 2, wherein the antiviral agent is present in a therapeuticallyeffective amount.
 4. The pharmaceutical composition of claim 3, whereinthe therapeutically effective amount is sufficient to provide the agentat a concentration of at least about 10 μM at a site of viral infectionin a subject.
 5. The pharmaceutical composition of claim 3, wherein thetherapeutically effective amount is a nontoxic amount.
 6. Thepharmaceutical composition of claim 3, wherein the therapeuticallyeffective amount is sufficient to provide the agent at a dosage/bodymass concentration of up to an amount selected from: 0.05, 0.1, 0.15,0.2, 0.3, 0.5, 1, 1.5, 2, 3, 5, 10, 15, 20, 30 mg/kg, about any of theforegoing values, and a range between any of the foregoing values. 7.The pharmaceutical composition of claim 2, wherein the pharmaceuticalcomposition is formulated to deliver the antiviral agent to: acirculatory system, a placenta, a fetus, an eye, a kidney, a brain, askin, one or more testes, one or more neurons, one or more stem cells, avagina, a spleen, an auditory system, or any combination of theforegoing, of a subject.
 8. A method of treatment or prevention of adisease mediated by the Zika virus (ZIKV) in a subject in need thereof,the method comprising administering to the subject a therapeuticallyeffective amount of the pharmaceutical composition of claim
 2. 9. Theantiviral agent of claim 1, wherein when the sequence comprises 5′-CATGAC CAG AAA CTC TCG TTT CCA A-3′ (SEQ ID NO: 3), the antiviral agenttargets a sequence in the 5′ region of the ZIKV virus.
 10. The antiviralagent of claim 1, wherein when the sequence comprises 5′-CAT GAC CAG AAACTC TCG TTT CCA A-3′ (SEO ID NO: 3), the sequence hybridizes underphysiological conditions with RNA containing the sequence 5′-TTG GAA ACGAGA GTT TCT GGT CAT G-3′ (SEQ ID NO: 2).
 11. The antiviral agent ofclaim 1, wherein when the sequence comprises 5′-CAT GGA GTC TCT GGT CTTTCC CAG C-3′ (SEQ ID NO: 5), the antiviral agent targets a sequence inthe 3′ region of the ZIKV virus.
 12. The antiviral agent of claim 1,wherein when the sequence comprises 5′-CAT GGA GTC TCT GGT CTT TCC CAGC-3′ (SEQ ID NO: 5) the sequence hybridizes under physiologicalconditions with RNA containing the sequence 5′-GCT GGG AAA GAC CAG AGACTC CAT G-3′ (SEQ ID NO: 4).
 13. The antiviral agent of claim 1, whereinthe agent comprises a moiety for intracellular delivery.
 14. Theantiviral agent of claim 1, wherein the agent comprises anocta-guanidine dendrimer delivery moiety.
 15. The antiviral agent ofclaim 1, wherein the agent comprises an octa-guanidine dendrimer of thefollowing structure:


16. The pharmaceutical composition of claim 2, wherein the diseasemediated by ZIKV is selected from the group consisting of: Zika fever,Guillain-Barre syndrome, a congenital defect, microcephaly, oculardisease, and Zika associated organ pathology.
 17. The pharmaceuticalcomposition of claim 3, wherein the therapeutically effective amount issufficient to provide the agent at a concentration of below an LD50 fora subject.
 18. The antiviral agent of claim 9, wherein the 5′ region ofthe ZIKV virus comprises a 5′ untranslated region.
 19. The antiviralagent of claim 11 wherein the 3′ region of the ZIKV virus comprises a 3′untranslated region.
 20. The antiviral agent of claim 11, wherein the 3′region of the ZIKV virus comprises a 3′ short hairpin structure.